JP2019532920A - MHC-binding peptide array and method of use thereof - Google Patents

Abstract

The present disclosure provides a composition comprising at least one association comprising a peptide and a major histocompatibility complex (MHC), wherein the peptide is an essential component of MHC and the peptide is linked to its C-terminus via a linker. In which the peptide is synthesized on the surface. In certain embodiments, the composition comprises a plurality of aggregates in a spatially ordered array. The present disclosure provides methods for making and using these compositions.

Description

  [01] The present disclosure is directed to molecular biology, synthetic biology, protein arrays and immunology, and medical and research tools.

  [02] For surface-bound peptides presented by appropriately assembled major histocompatibility complex (MHC) that can be synthesized in situ for in vitro analysis of peptides in the correct biological context. There has been a long-awaited need in the technical field that has not been met. The present disclosure provides a solution to this long-awaited but unmet need.

  [03] The present disclosure assesses the interaction between peptides presented by associated major histocompatibility complex (MHC) and immune cells (eg, T cells) and biologically in vivo. In vitro systems are provided for identifying related peptides and cells. The compositions of the present disclosure provide the unique ability to identify a single peptide that is immunodominant in vivo by providing a surface with each peptide bound to the surface in a spatially ordered array. The spatial order of the peptides is made possible by in situ synthesis of each peptide on the surface.

  [04] The success of the composition of the present disclosure is not only the failure of others to try, but also opposes the dominant opinion in the state of the art. For example, for compositions comprising class I MHC, the dominant knowledge is that both the amino terminus (N terminus) and carboxy terminus (C terminus) of the peptide are free, unbound, Or indicate that it must be moored.

[05] The compositions of the present disclosure can be used to identify immunodominant peptides for any condition. Furthermore, the state of the art relies on computer algorithms to predict immunodominant peptides that may or may not be related in vivo. In contrast, the compositions of the present disclosure have the ability to simultaneously analyze at least 10 ⁶ unique peptides to specifically identify unique peptides that activate immune cells. Since the MHC on the surface accurately reproduces the peptide-MHC aggregate so that the peptide-MHC aggregate appears on the surface of the cell in vivo on the surface of the array, Identify related peptides in vivo, thus eliminating the need for further experimental validation and eliminating time-consuming and expensive steps required by existing technologies. Since each of these peptides is spatially ordered and their sequence is pre-determined, the sequence of the immunodominant peptide is readily known and required by the existing technology of sequencing related peptides. It also eliminates another time consuming and expensive process.

  [06] By analyzing peptide variants, the compositions of the present disclosure can be used for successful association of each peptide-MHC, regardless of class (eg, MHC class I or II) and which leukocyte antigen (eg, HLA). Regardless of whether -A, HLA-B or HLA-C) contributes to MHC, it can be used to identify essential amino acids (also called "anchor points").

  [07] Specifically, the disclosure provides a composition comprising at least one association comprising a peptide and a major histocompatibility complex (MHC), wherein the peptide is an essential component of MHC, Compositions are provided that are attached to the surface at their C-terminus via a linker and the peptide is synthesized on the surface. In certain embodiments, the at least one aggregate is a plurality of aggregates.

  [08] MHC includes humans (ie, human leukocyte antigen (HLA) genes), primates (eg, apes, monkeys, chimpanzees, and bonobos), and mice (ie, mouse leukocyte antigen (MLA) genes). It may be encoded by a leukocyte antigen gene from any mammalian species that is not limited thereto.

  [09] In certain embodiments of the disclosed compositions, the MHC is a class I MHC.

[010] In certain embodiments of the compositions of the present disclosure, wherein the MHC is a Class I MHC, the alpha chain of the MHC may be cleaved. For example, in certain embodiments, the α chain of MHC does not include a transmembrane or cytoplasmic region. In certain embodiments in which the MHC is a class I MHC, the MHC alpha chain does not include a hinge region. In certain embodiments, the MHC is a class I MHC, the alpha chain of the MHC does not include a transmembrane region, hinge region or cytoplasmic region. In certain embodiments where the MHC is a class I MHC, the α chain of the MHC comprises an α ₁ domain, an α ₂ domain, and an α ₃ domain. In certain embodiments where the MHC is a class I MHC, the alpha chain of the MHC is HLA-A gene, HLA-B gene, HLA-C gene, HLA-E gene, HLA-F gene, HLA-G gene, HLA -Can be encoded by a sequence derived from an HLA gene selected from the group consisting of a K pseudogene and an HLA-L pseudogene. Alternatively, if the MHC is a class I MHC, the MHC alpha chain is encoded by a sequence derived from an HLA gene selected from the group consisting of an HLA-A gene, an HLA-B gene and an HLA-C gene. obtain. Alternatively, if the MHC is a class I MHC, the alpha chain of the MHC can be encoded by a sequence derived from the HLA-A gene.

[011] In certain embodiments of the compositions of the present disclosure, the MHC is a class I MHC. The alpha chain of MHC can be encoded by a sequence derived from the HLA-A gene, particularly HLA-A ^* 11 ^: 01. The amino acid sequence of ^HLA-A * 11:01 can consist or a EmujiesueichiesuemuaruwaiefuwaitiesubuiesuaruPijiarujiiPiaruefuaieibuijiwaibuididitikyuefubuiaruefudiesudieieiesukyuaruemuiPiarueiPidaburyuaiikyuiJipiiwaidaburyudikyuitiaruenubuikeieikyuesukyutidiarubuidierujitieruarujiwaiwaienukyuesuidijiesueichitiaikyuaiemuwaijishidibuiJipidijiaruefueruarujiwaiarukyudieiwaidijikeidiwaiaieieruenuidieruaruesudaburyutieieidiemueieikyuaitikeiarukeidaburyuieieieichieieiikyukyuarueiwaieruijiarushibuiidaburyueruaruaruwaieruienujikeiitierukyuarutidiPiPikeitieichiemutieichieichiPiaiesudieichiieitieruarushidaburyueierujiefuwaiPieiiaitierutidaburyukyuarudijiidikyutikyuditiierubuiitiaruPieijidijitiefukyukeidaburyueieibuibuibuiPiSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO: 1).

[012] In certain embodiments of the compositions of the present disclosure, the MHC is a class I MHC. The α chain of MHC can be encoded by sequences derived from the HLA-B gene, particularly HLA-B ^* 07 ^: 02. The amino acid sequence of ^HLA-B * 07:02 can consist or a JiesueichiesuemuaruwaiefuwaitiesubuiesuaruPijiarujiiPiaruefuaiesubuijiwaibuididitikyuefubuiaruefudiesudieiEiesupiaruiiPiarueiPidaburyuaiikyuiJipiiwaidaburyudiaruenutikyuaiwaikeieikyueikyutidiaruiesueruaruenueruarujiwaiwaienukyuesuieijiesueichitierukyuesuemuwaijishidibuiJipidijiaruerueruarujieichidikyuwaieiwaidijikeidiwaiaieieruenuidieruaruesudaburyutieieiditieieikyuaitikyuarukeidaburyuieieiaruieiikyuaruarueiwaieruijiishibuiidaburyueruaruaruwaieruienujikeidikeieruiarueidiPiPikeitieichibuitieichieichiPiaiesudieichiieitieruarushidaburyueierujiefuwaiPieiiaitierutidaburyukyuarudijiidikyutikyuditiierubuiitiaruPieijidiarutiefukyukeidaburyueieibuibuibuiPiSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO: 2).

[013] In certain embodiments of the compositions of the present disclosure, the MHC is a class I MHC. The α chain of MHC can be encoded by sequences derived from the HLA-C gene, in particular HLA-C ^* 07 ^: 02. The amino acid sequence of ^HLA-C * 07:02 can consist or a EsueichiesuemuaruwaiefuditieibuiesuaruPijiarujiiPiaruefuaiesubuijiwaibuididitikyuefubuiaruefudiesudieiEiesupiarujiiPiarueiPidaburyubuiikyuiJipiiwaidaburyudiaruitikyukeiwaikeiarukyueikyueidiarubuiesueruaruenueruarujiwaiwaienukyuesuidijiesueichitierukyuaruemuesujishidieruJipidijiaruerueruarujiwaidikyuesueiwaidijikeidiwaiaieieruenuidieruaruesudaburyutieieiditieieikyuaitikyuarukeieruieieiarueieiikyueruarueiwaieruijitishibuiidaburyueruaruaruwaieruienujikeiitierukyuarueiiPiPikeitieichibuitieichieichiPieruesudieichiieitieruarushidaburyueierujiefuwaiPieiiaitierutidaburyukyuarudijiidikyutikyuditiierubuiitiaruPieijidijitiefukyukeidaburyueieibuibuibuiPiSGQEQRYTCHMQHEGLQEPLTLSWE (SEQ ID NO: 3).

[014] In certain embodiments of the disclosed compositions, the HLA-A ^* 11: 01, HLA-B ^* 07: 02 and HLA-C ^* 07 ^: 02 amino acid sequences from the UniProt database are cleaved to yield C Hinge, transmembrane and cytoplasmic regions were removed at the ends, and leader peptide sequences were removed from the N-terminus.

  [015] In certain embodiments of the compositions of the present disclosure, the MHC is a Class II MHC.

[016] In certain embodiments of the disclosed compositions wherein the MHC is a Class II MHC, the alpha chain of the MHC may be cleaved. For example, in certain embodiments, the α chain of MHC does not include a transmembrane or cytoplasmic region. In certain embodiments where the MHC is a class II MHC, the α chain of the MHC comprises an α ₁ domain and an α ₂ domain. In certain embodiments where the MHC is a class II MHC, the alpha chain of the MHC is selected from the group consisting of an HLA-DM gene, an HLA-DO gene, an HLA-DP gene, an HLA-DQ gene, and an HLA-DR gene. It can be encoded by a sequence derived from the HLA gene. In certain embodiments in which the MHC is a class II MHC, the MHC β chain is cleaved. For example, in certain embodiments, the MHC β chain does not include a transmembrane or cytoplasmic region. In certain embodiments where the MHC is a class II MHC, the MHC β chain comprises a β ₁ domain and a β ₂ domain. In certain embodiments where the MHC is a class II MHC, the MHC β chain is selected from the group consisting of an HLA-DM gene, an HLA-DO gene, an HLA-DP gene, an HLA-DQ gene, and an HLA-DR gene. It can be encoded by a sequence derived from the HLA gene.

  [017] In certain embodiments of the compositions of the present disclosure, the MHC and / or at least one aggregate comprises a carrier molecule. The carrier molecules of the present disclosure facilitate the integration of MHC components with each peptide on the surface to form at least one peptide-MHC aggregate. Although there are many mechanisms by which this promotion can be achieved, in certain embodiments, the carrier molecule of the present disclosure can be one or more of the one or more components of the MHC before contacting the peptide on the surface. Can be combined with ingredients. A carrier molecule that binds to one or more components of the MHC before it contacts the peptide on the surface is essential for the peptide to bind to one or more components of the MHC. It does not bind to the residue and / or binding site of one or more components. The carrier molecule may remain bound to the fully associated MHC on the surface, or may dissociate after the components of the MHC have associated with the peptide on the surface. The carrier molecules of the present disclosure do not alter the response of T cells to peptides presented by MHC on the surface.

  [018] In certain embodiments of the disclosed compositions, the carrier molecule comprises, consists essentially of, or can consist of bovine serum albumin (BSA). In certain embodiments where the MHC is a class I MHC, the carrier molecule comprises, consists essentially of, or can consist of bovine serum albumin (BSA).

  [019] In certain embodiments of the disclosed compositions, the linker comprises hexanoic acid. In certain embodiments, the linker may comprise 1 to 5 monomer units. In certain embodiments, the linker may comprise 3-5 monomer units. In certain embodiments, the linker may comprise at least one negatively charged monomer unit. For example, the at least one negatively charged monomer unit can include a negatively charged amino acid. The negatively charged amino acid may be aspartic acid (D) or glutamic acid (E). In certain embodiments, the linker may comprise hexanoic acid and at least one negatively charged amino acid having a length of 5 monomers. Examples of linkers include, but are not limited to, (surface) -5HEX, (surface) -HEX-E-3HEX, (surface) -HEX-D-3HEX, (surface) -2HEX-E-HEX. .

  [020] In certain embodiments of the disclosed compositions, the linker comprises polyethylene glycol (PEG). In certain embodiments, the linker may comprise 1 to 5 monomer units. In certain embodiments, the linker may comprise 3-5 monomer units. In certain embodiments, the linker may comprise at least one negatively charged monomer unit. For example, the at least one negatively charged monomer unit can include a negatively charged amino acid. The negatively charged amino acid may be aspartic acid (D) or glutamic acid (E).

  [021] In certain embodiments of the disclosed compositions, the linker comprises a mixture of glycine (G) and serine (S) amino acids. For example, the linker may comprise a 3: 1 ratio mixture of glycine (G): serine (S) amino acids. In certain embodiments, the linker may comprise 1 to 5 monomer units. In certain embodiments, the linker may comprise 3-5 monomer units. In certain embodiments, the linker may comprise at least one negatively charged monomer unit. For example, the at least one negatively charged monomer unit can include a negatively charged amino acid. The negatively charged amino acid may be aspartic acid (D) or glutamic acid (E).

  [022] In certain embodiments of the compositions of the present disclosure, the linker comprises at least one negatively charged monomer unit, and the aggregate comprises MHC encoded by the HLA2 gene. In certain embodiments, the linker comprises at least one negatively charged monomer unit, the linker consists of 3-5 monomers, and the aggregate comprises MHC encoded by the HLA2 gene. In certain embodiments, the linker comprises at least one negatively charged monomer unit, the linker consists of 3 monomers, and the aggregate comprises MHC encoded by the HLA2 gene. In certain embodiments, the linker comprises at least one negatively charged monomer unit, the linker consists of 5 monomers, and the aggregate comprises MHC encoded by the HLA2 gene.

  [023] In certain embodiments of the compositions of the present disclosure, each peptide of the at least one aggregate or plurality of aggregates comprises, consists essentially of, or consists of 6-30 amino acids including endpoints. Can be. In certain embodiments of the compositions of the present disclosure, each peptide of the at least one aggregate or plurality of aggregates comprises, consists essentially of, or consists of 6-20 amino acids including endpoints. it can. In certain embodiments of the compositions of the present disclosure, each peptide of the at least one aggregate or plurality of aggregates comprises, consists essentially of, or consists of 6-12 amino acids including endpoints. it can. In certain embodiments of the disclosed compositions wherein the MHC is a Class I MHC, it comprises, consists essentially of, or consists of 6-12 amino acids including endpoints. In certain embodiments of the compositions of the present disclosure, each peptide of the at least one aggregate or plurality of aggregates comprises, consists essentially of, or consists of 9 amino acids. In certain embodiments of the disclosed compositions wherein the MHC is a Class I MHC, each peptide of the at least one aggregate or plurality of aggregates comprises, consists essentially of, or consists of 9 amino acids be able to. In certain embodiments of the compositions of the present disclosure, each peptide of the at least one aggregate or plurality of aggregates comprises, consists essentially of, or consists of 12-30 amino acids including endpoints. it can. In certain embodiments of the disclosed compositions wherein the MHC is a Class II MHC, each peptide of the at least one association or plurality of associations comprises 12-30 amino acids including endpoints, and then consists essentially of Can consist of or consist of.

  [024] In certain embodiments of the compositions of the present disclosure, at least one aggregate peptide is synthesized in situ using a digital micromirror device (DMD). In certain embodiments of the disclosed compositions, the multi-aggregate peptide is synthesized in situ using a digital micromirror device (DMD). In certain embodiments, the DMD includes at least one micromirror. In certain embodiments, the DMD includes a plurality of micromirrors. In certain embodiments, each micromirror corresponds to a surface microregion, and the micromirror corresponding to the microregion is directed to the synthesis of each peptide in the microregion. In certain embodiments, the surface includes a plurality of microregions, where the number of micromirrors is equal to the number of microregions. As used herein, the term “microarea” is meant to describe a virtual boundary rather than a physical boundary that exists on the surface itself. Although micromirrors can direct the synthesis of peptides at any site on the surface, the microregions affected by the micromirror are preferably placed on the surface so that they are aligned perpendicular to the micromirror. Thus, the microregions affected by the micromirror can be within a few millimeters or centimeters from an array directly below the micromirror or perpendicular to the edge of the micromirror.

[025] In certain embodiments of the disclosed compositions, each microregion is 10 ³ to 10 ⁸ , particularly in embodiments where the peptide is synthesized in situ using a digital micromirror device (DMD). Peptides can be included. In certain embodiments, the surface can have a density of 1.24 × 10 ¹³ amines per cm ² , where the amine is the first functional group attached to the surface relative to later Linkers and peptides are attached during in situ synthesis. When the surface has a 1 cm ² per 1.24 × ^{10 13} pieces of amine density, surface can include at least ¹⁰⁶ peptides, which corresponds to at least ¹⁰⁶ peptides -MHC aggregate. When the surface has a density of 1.24 × 10 ¹³ amines per cm ² , the surface can contain 10 ³ to 10 ⁸ “unique” peptides per micromirror. When the surface has a density of 1.24 × 10 ¹³ amines per cm ² , the surface is 10 ³ to 10 ⁸ “unique” per microregion, where each microregion corresponds to one micromirror. Peptides can be included. The term “unique peptide” is meant to describe a unique peptide sequence. In addition to a unique peptide, each micromirror has a replicating peptide, in other words, has the same sequence and directs the micromirror to synthesize a peptide having a unique sequence among peptides on the same surface or section of the same surface. Multiple peptides can be generated that are generated by a unique program or feature (a set of instructions).

  [026] In certain embodiments of the compositions of the present disclosure, particularly in embodiments where the peptide is synthesized in situ using a digital micromirror device (DMD), the first microregion is each second or At least a first peptide having a unique amino acid sequence when compared to the amino acid sequence of at least a second or next peptide in the next microregion. In certain embodiments, the first microregion comprising at least one peptide having a unique amino acid sequence further comprises at least one copy of a peptide having a unique amino acid sequence. For example, the second or next microregion may be juxtaposed with the first microregion. The juxtaposition means that the virtual boundaries of the first and second microregions are physically juxtaposed (for example, the microregions are adjacent to each other). In certain embodiments, the first microregion is unique when compared to the amino acid sequence of at least a second or next peptide within each second or next microregion juxtaposed to the first microregion. At least a first peptide having an amino acid sequence. In certain embodiments, the first microregion has a unique amino acid sequence when compared to the amino acid sequence of at least a second or next peptide in each second or next microregion on the surface. At least.

[027] In certain embodiments of the compositions of the present disclosure, in embodiments, particularly peptides are synthesized in situ using a digital micromirror device (DMD), the surface is 1.24 × 10 ¹³ cells per square centimeter Of peptides.

  [028] In certain embodiments of the compositions of the present disclosure, the surface comprises two or more compartments. In certain embodiments of the compositions of the present disclosure, the surface comprises 2-48 compartments including endpoints.

  [029] In certain embodiments of the compositions of the present disclosure, the composition further comprises at least one T cell. In certain embodiments of the compositions of the present disclosure, the composition further comprises at least one T cell per compartment. Optionally, at least one T cell can bind to the surface.

  [030] In certain embodiments, the compositions of the present disclosure are detectable to recognize molecules released from at least one T cell upon activation of at least one T cell by at least one peptide on the surface. It may further contain other drugs. The detectable agent can be any organic or inorganic molecule that has a detectable label that, upon activation, can specifically bind to at least one molecule released from at least one T cell. In certain embodiments, the detectable agent is an antibody. Although the molecule released from at least one T cell can be any molecule of the cell's secretome, in certain embodiments, the molecule released from at least one T cell is a cytokine. Examples of cytokines include interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), and interleukin 6 (IL-6). , Granulocyte macrophage colony stimulating factor (GM-CSF) and interferon-gamma (INFγ).

  [031] In certain embodiments, the compositions of the present disclosure may include at least two surfaces. The surface of the composition of the present disclosure may be flat. For example, the surface of the composition of the present disclosure may be a chip, plate, or slide.

  [032] In certain embodiments, the compositions of the present disclosure may include at least two surfaces. The surface of the composition of the present disclosure may be curved, convex, or concave. In certain embodiments, the surface is a bead.

  [033] In certain embodiments of the disclosed compositions, each peptide of the plurality of aggregates is synthesized on the surface to produce a spatially ordered array. In the context of synthesis using DMD technology, each micromirror has a unique program for reflecting light on the surface or for polarizing light from the surface. The peptides on the surface of the present disclosure may be synthesized using photoprotected amino acids. When the micromirror directs light to the surface, the last amino acid added to the growing peptide is deprotected and becomes available for binding to another amino acid. By providing each micromirror with a unique sequence movement (also called “job”), the DMD can synthesize at least one unique peptide sequence corresponding to each micromirror. By preprogramming the mirror in a predetermined pattern, the sequence of each peptide synthesized on the surface is known by its relative position with respect to the micromirror used to synthesize it.

  [034] In certain embodiments, the surface is a chip and three chips are placed on a slide for simultaneous manufacture and use. In certain embodiments, the spatial arrangement of multiple peptides for each of these three chips can be the same so as to provide experimental replicates. In certain embodiments, the spatial arrangement of multiple peptides on each of these three chips can be defined to provide a higher throughput assay.

  [035] The present disclosure is a method of making a composition of the present disclosure, wherein a surface comprising at least one peptide and an MHC composition are suitable for generating at least one peptide-MHC aggregate. Contacting the peptide under conditions, wherein the peptide is an essential component of the aggregate, at least one peptide is attached to the surface at its C-terminus via a linker, and at least one peptide is synthesized on the surface Provide a method. In certain embodiments, the at least one aggregate is a plurality of aggregates.

  [036] In certain embodiments of the methods of making the disclosed compositions, the surface may be treated with a binding buffer prior to the contacting step. In certain embodiments, the binding buffer comprises 1% casein, 10 mM pH 7.4 Tris, and 0.25% Tween. Optionally, the binding buffer may further comprise a blocking agent.

  [037] In certain embodiments of the methods of making the compositions of the present disclosure, the surface and the MHC composition remain in contact for 8-24 hours including the endpoints. In a particular embodiment of the method of making the composition of the present disclosure, the surface and the MHC composition remain in contact for about 12 hours. The contacting step can be performed at room temperature. Alternatively, the contacting step can be performed at 4 ° C.

  [038] In a particular embodiment of the method of making a composition of the present disclosure, the MHC composition comprises BSA, solubilized β2-microglobulin (β2m) and solubilized α chain. In certain embodiments, the MHC composition comprises solubilized α chain: solubilized β2m in a mole: molar ratio of 1: 1 to 1: 2 including endpoints. The solubilized alpha chain can be encoded by the HLA-A gene, the HLA-B gene or the HLA-C gene. In certain embodiments, the solubilized alpha chain comprises: (a) back-translating an amino acid sequence encoding the solubilized alpha chain into a codon optimized deoxyribonucleic acid (DNA) sequence; (b) a codon optimized deoxyribonucleic acid ( DNA) synthesizing the sequence as a double-stranded DNA molecule, (c) expressing the double-stranded DNA molecule in the form of an inclusion body to produce an α chain, and (d) a step of solubilizing the α chain. It is produced by the method of including. In certain embodiments of the method of making a solubilized alpha chain, the codon optimized deoxyribonucleic acid (DNA) sequence is optimized for expression in E. coli. In certain embodiments of the method of making a solubilized alpha chain, the expression step comprises cloning a double stranded DNA molecule into an expression vector, wherein optionally the expression vector is a plasmid.

  [039] In certain embodiments of the methods of making the compositions of the present disclosure, the MHC composition comprises a carrier molecule, a solubilized beta chain, and a solubilized alpha chain. In certain embodiments where the MHC is a class II MHC, the carrier molecule can be an invariant chain, CLIP, CD74 peptide or a functional fragment thereof. In certain embodiments where the MHC is a class II MHC, the carrier molecule can be BSA.

  [040] In a particular embodiment of the method of making the composition of the present disclosure, the MHC composition comprises 0.1% to 10% BSA including endpoints. The MHC composition can include 1% to 5% BSA including endpoints. The MHC composition can include 2% to 3% BSA including endpoints. Optionally, BSA is formulated in a BSA buffer. In certain embodiments, the BSA buffer comprises 20 mM Tris-HCl pH 7.8.

  [041] In certain embodiments of the methods of making the composition of the present disclosure, the MHC composition may further comprise a buffer. The MHC buffer can contain 10 mM Tris-HCl pH 8.5.

  [042] In a particular embodiment of the method of making a composition of the present disclosure, prior to contacting the surface, the MHC composition comprises (a) incubating the MHC composition at 4 ° C overnight; (b) Produced by a method comprising separating the precipitate from the MHC composition, and (c) collecting the precipitate-free MHC composition for contact with the surface. In certain embodiments, the separation step can include a centrifugation step, wherein the MHC composition that is free of precipitate is the supernatant resulting from centrifugation. In certain embodiments of the separation step, the recovery step further comprises the step of concentrating the MHC composition that is free of precipitate. In certain embodiments, the collecting step further comprises filtering the MHC composition free of precipitate.

  [043] The present disclosure provides the use of the compositions of the present disclosure for the identification of one or more peptide antigens that are immunodominant when presented in vivo by MHC. For example, the present disclosure provides the use of a composition of the present disclosure for the identification of one or more peptide antigens that are immunodominant when presented in vivo by an MHC, comprising: (a) at least one T Contacting the cell with the composition; (b) upon activation of the T cell, detecting at least one molecule secreted from the at least one T cell, and thereby presented in vivo by the MHC Uses are provided that include identifying one or more peptide antigens that are immunodominant. In a particular embodiment of this use, the immunodominant peptide antigen is immunogenic.

  [044] In a particular embodiment of the use of the composition of the present disclosure for identification of one or more peptide antigens that are immunodominant when presented in vivo by MHC, the MHC is a class I MHC .

  [045] In a specific embodiment of the use of the composition of the present disclosure for the identification of one or more peptide antigens that are immunodominant when presented in vivo by an MHC, the MHC is a class II MHC .

  [046] In a specific embodiment of the use of the composition of the present disclosure for the identification of one or more peptide antigens that are immunodominant when presented in vivo by MHC, the peptide antigen is a neoantigen . In certain embodiments, the neoantigen may comprise one or more amino acids essential for binding to MHC. In certain embodiments, one or more amino acids of a neoantigen that are not essential for binding to MHC (“non-essential” amino acids) have one or more amino acid sequences compared to the wild-type sequence of the peptide. Substitution may be included.

  [047] In a specific embodiment of the use of the composition of the present disclosure for the identification of one or more peptide antigens that are immunodominant when presented in vivo by MHC, the peptide antigen is an autoantigen. In certain embodiments, the self-antigen stimulates T cells and induces an autoimmune response.

  [048] The present disclosure provides a self-antigen of the present disclosure that is identified as immunodominant when presented in vivo by an MHC, for the manufacture of a medicament that reduces or prevents an autoimmune response, complementary to the self-antigen There is provided the use of a sequence, an antibody that specifically binds to the self-antigen, or a chimeric antigen receptor that specifically binds to the self-antigen.

  [049] In certain embodiments of the use of the composition of the present disclosure for identification of one or more peptide antigens that are immunodominant when presented in vivo by MHC, the peptide antigen is a cancer antigen. is there. In certain embodiments, the cancer antigen stimulates T cells and induces an immune response. T cells may be genetically modified. For example, T cells may include genetically modified T cell receptors that specifically bind to cancer antigens. In certain embodiments, the T cell may comprise a chimeric antigen receptor that specifically binds to a cancer antigen.

  [050] The present disclosure provides a vaccine comprising a cancer antigen of the present disclosure identified as immunodominant when presented in vivo by MHC.

  [051] The present disclosure relates to a cancer antigen of the present disclosure that is identified as immunodominant when presented in vivo by an MHC, for the manufacture of a medicament that enhances or induces an immune response, complementary to the cancer antigen Use of a specific antigen sequence, an antibody that specifically binds to the cancer antigen, or a chimeric antigen receptor that specifically binds to the cancer antigen.

  [052] In a particular embodiment of the use of the composition of the present disclosure for the identification of one or more peptide antigens that are immunodominant when presented in vivo by MHC, the peptide antigens are bacteria, viruses, Or a microbial antigen.

  [053] In a particular embodiment of the use of the composition of the present disclosure for the identification of one or more peptide antigens that are immunodominant when presented in vivo by MHC, the peptide antigen is a viral antigen; Here, viral antigens stimulate T cells and induce an immune response. In certain embodiments, the viral antigen is derived from cytomegalovirus (CMV). T cells may be genetically modified. For example, T cells may include genetically modified T cell receptors that specifically bind to viral antigens. In certain embodiments, T cells may comprise a chimeric antigen receptor that specifically binds to a viral antigen.

  [054] The present disclosure provides a vaccine comprising a viral antigen of the present disclosure identified as immunodominant when presented in vivo by MHC. In certain embodiments, the viral antigen is derived from cytomegalovirus (CMV).

  [055] The present disclosure provides a viral antigen of the present disclosure identified as immunodominant when presented in vivo by an MHC for the manufacture of a medicament that enhances or induces an immune response, a sequence complementary to the viral antigen Use of an antibody that specifically binds to the viral antigen, or a chimeric antigen receptor that specifically binds to the viral antigen. In certain embodiments, the viral antigen is derived from cytomegalovirus (CMV).

  [056] In a particular embodiment of the use of the composition of the present disclosure for the identification of one or more peptide antigens that are immunodominant when presented in vivo by MHC, the peptide antigen is a bacterial antigen; Here bacterial antigens stimulate T cells and induce an immune response. T cells may be genetically modified. For example, T cells may include genetically modified T cell receptors that specifically bind to bacterial antigens. In certain embodiments, the T cell may comprise a chimeric antigen receptor that specifically binds to a bacterial antigen.

  [057] The present disclosure provides a vaccine comprising a bacterial antigen of the present disclosure identified as immunodominant when presented in vivo by MHC.

  [058] The present disclosure provides a bacterial antigen of the present disclosure identified as immunodominant when presented in vivo by an MHC for the manufacture of a medicament that enhances or induces an immune response, a sequence complementary to the bacterial antigen Use of an antibody that specifically binds to the bacterial antigen, or a chimeric antigen receptor that specifically binds to the bacterial antigen.

  [059] In a particular embodiment of the use of the composition of the present disclosure for the identification of one or more peptide antigens that are immunodominant when presented in vivo by MHC, the peptide antigen is a microbial antigen; Here, microbial antigens stimulate T cells and induce an immune response. T cells may be genetically modified. For example, T cells may include genetically modified T cell receptors that specifically bind to microbial antigens. In certain embodiments, the T cell may comprise a chimeric antigen receptor that specifically binds to a microbial antigen.

  [060] The present disclosure provides a vaccine comprising a microbial antigen of the present disclosure identified as immunodominant when presented in vivo by MHC.

  [061] The present disclosure provides a microbial antigen of the present disclosure identified as immunodominant when presented in vivo by an MHC for the manufacture of a medicament that enhances or induces an immune response, a sequence complementary to the microbial antigen Use of an antibody that specifically binds to the microbial antigen, or a chimeric antigen receptor that specifically binds to the microbial antigen.

  [062] The disclosure includes (a) contacting at least one T cell with the composition; (b) detecting at least one molecule secreted from the at least one T cell upon activation; And (c) providing the use of a composition of the present disclosure for the identification of at least one T cell stimulated by at least one peptide comprising imaging at least one T cell.

  [063] In certain embodiments of the use of the composition of the present disclosure for the identification of at least one T cell stimulated by at least one peptide, the at least one T cell is not genetically modified.

  [064] In certain embodiments of the use of the composition of the present disclosure for the identification of at least one T cell stimulated by at least one peptide, the at least one T cell is genetically modified. For example, in certain embodiments, the at least one T cell may comprise a modified T cell receptor that specifically binds to at least one peptide. In certain embodiments, at least one T cell comprises a chimeric antigen receptor (CAR) that specifically binds to at least one peptide.

  [065] In certain embodiments of the use of the composition of the present disclosure for the identification of at least one T cell stimulated by at least one peptide, the imaging step comprises at least one T cell, one or Contacting with one or more detectable agents that recognize a plurality of cell surface markers. The detectable agent can be any organic or inorganic molecule that has a detectable label that, upon activation, can specifically bind to at least one molecule released from at least one T cell. In certain embodiments, the detectable agent is an antibody. Examples of cell surface markers include, but are not limited to CD34, CD45, CD15, CD14, CD3, CD19, CD61, CD4, CD8 and CD25. For example, stem cells can be identified by the expression of CD34. Granulocytes can be identified by the expression of CD45 and CD15. Monocytes can be identified by the expression of CD45 and CD14. T cells (also called T lymphocytes) can be identified by the expression of CD45 and CD3. B cells (also called B lymphocytes) can be identified by the expression of CD45 and CD19. Platelets can be identified by the expression of CD45 and CD61. Among T cells, helper T cells can be identified by the expression of CD45, CD3 and CD4. Among T cells, cytotoxic T cells can be identified by the expression of CD45, CD3 and CD8. Among T cells, activated T cells can be identified by the expression of CD45, CD3 and CD25.

  [066] In certain embodiments of the compositions of the present disclosure for identification of at least one T cell stimulated by at least one peptide, at least one T cell may be included in the cell population. A cell population can be isolated from any biological fluid including, but not limited to, whole blood, serum, plasma, peripheral blood, umbilical cord blood, lymph, cerebrospinal fluid (CSF), and amniotic fluid. A cell population can be isolated from any living tissue, including but not limited to lymphoid tissue, bone marrow, tumor tissue, and biopsy tissue. The cell population may be uniform or heterogeneous. A cell population can comprise, consist essentially of, or consist of immune cells or T cells. The cell population can comprise, consist essentially of, or consist of a mixture of helper T cells and cytotoxic T cells prior to contacting the composition of the present disclosure. In certain embodiments, the cell population does not contain any activated T cells prior to contacting the composition of the present disclosure. Accordingly, the present invention provides a composition comprising at least one association or a plurality of associations comprising a peptide and a major histocompatibility complex (MHC), wherein the peptide is an essential component of MHC, Compositions are provided that are attached to the surface at their C-terminus via a linker and the peptide is synthesized on the surface.

  [067] The MHC can be a Class I or Class II MHC. The MHC can include a carrier molecule that can be bovine serum albumin (BSA). The linker can comprise a 3: 1 ratio mixture of hexanoic acid, polyethylene glycol (PEG), or glycine (G): serine (S) amino acids. The linker can comprise 1 to 5 monomer units, preferably 3 to 5 monomer units. The linker can comprise at least one negatively charged monomer unit, which can be a negatively charged amino acid such as aspartic acid (D) or glutamic acid (E). In one embodiment, the linker comprises hexanoic acid and at least one aspartic acid (D) or glutamic acid (E), preferably within 5 monomer units.

  [068] MHC is a human leukocyte antigen 2 (HLA2) gene, another human leukocyte antigen (HLA) gene, a mammalian leukocyte antigen gene, a primate leukocyte antigen gene, a mouse leukocyte antigen (MLA) gene, or It can be encoded by the human leukocyte antigen (HLA) gene, where the α chain of MHC is cleaved. In the latter case, the α chain does not contain a transmembrane or cytoplasmic region. The α chain may not include the hinge region. The α chain can include an α1 domain, an α2 domain, and an α3 domain. The α chain is selected from the group consisting of HLA-A gene, HLA-B gene, HLA-C gene, HLA-E gene, HLA-F gene, HLA-G gene, HLA-K pseudogene and HLA-L pseudogene Can be encoded by a sequence derived from the HLA gene. The alpha chain can be encoded by a sequence derived from an HLA gene selected from the group consisting of HLA-A gene, HLA-B gene and HLA-C gene. The α chain can also include an α1 domain and an α2 domain. In one embodiment, the α chain is a sequence derived from an HLA gene selected from the group consisting of HLA-DM gene, HLA-DO gene, HLA-DP gene, HLA-DQ gene and HLA-DR gene. Can be coded by

  [069] The MHC β chain may be cleaved, for example, not including the transmembrane or cytoplasmic region. The β chain can comprise a β1 domain and a β2 domain. The β chain can be encoded by a sequence derived from an HLA gene selected from the group consisting of HLA-DM gene, HLA-DO gene, HLA-DP gene, HLA-DQ gene and HLA-DR gene.

  [070] Each peptide of the plurality of aggregates can be composed of 6-30, preferably 6-20, more preferably 6-12, most preferably 9 amino acids, including endpoints. At least one aggregate peptide can be synthesized in situ using a digital micromirror device (DMD), where each micromirror corresponds to a microregion of the surface, and the micromirror corresponding to the microregion is , Directed to the synthesis of each peptide in the microregion.

  [071] In this context, the present invention is a method of making a composition as disclosed above, comprising a surface comprising at least one peptide, an MHC composition, and at least one peptide-MHC aggregate. The peptide is an essential component of MHC, the peptide is attached to the surface at its C-terminus via a linker, and the peptide is synthesized on the surface A method is also provided. Prior to the contacting step, the surface may be treated with a binding buffer, such as 1% casein, 10 mM pH 7.4 Tris, 0.25% Tween 20, and optionally a blocking reagent. . The surface and the MHC composition may remain in contact for 8-24 hours including the endpoint, for example about 12 hours, for example at room temperature or at 4 ° C. The MHC composition may comprise BSA, solubilized β2-microglobulin (β2m) and solubilized α chain. The composition may also comprise a carrier molecule, a solubilized β chain, and a solubilized α chain. The carrier molecule is an invariant chain, CLIP, CD74 peptide or functional fragment thereof, or bovine serum albumin (BSA), preferably 0.1% -10% BSA including endpoints, more preferably 1% -5% BSA, most preferably 2% to 3% BSA. BSA may be formulated, for example, in a BSA buffer containing 20 mM Tris-HCl pH 7.8. The MHC composition further comprises a buffer, eg, 10 mM Tris-HCl pH 8.5.

[072] Prior to contacting the surface, the MHC composition is
(A) incubating the MHC composition at 4 ° C. overnight;
Made by a method comprising (b) separating the precipitate from the MHC composition, and (c) collecting the precipitate-free MHC composition for contact with the surface.

[073] The separation step may further comprise a centrifugation step, wherein the precipitate free MHC composition is the supernatant resulting from centrifugation. The recovery step may further include a step of concentrating the MHC composition that does not include a precipitate. The recovery step may further include a step of filtering the MHC composition that does not include the precipitate. The MHC composition may comprise solubilized α chain: solubilized β2m in a molar: molar ratio of 1: 1 to 1: 2 including endpoints. The solubilized alpha chain can be encoded by the HLA-A gene, the HLA-B gene or the HLA-C gene. The solubilized α chain is
(A) back-translating an amino acid sequence encoding a solubilized α chain into a codon optimized deoxyribonucleic acid (DNA) sequence;
(B) synthesizing a codon optimized deoxyribonucleic acid (DNA) sequence as a double stranded DNA molecule;
It is produced by a method comprising the steps of (c) expressing a double-stranded DNA molecule in the form of an inclusion body to produce an α chain, and (d) solubilizing the α chain.

  [074] Optionally, the codon optimized deoxyribonucleic acid (DNA) sequence is optimized for expression in E. coli.

  [075] The composition disclosed above is for the identification of one or more peptide antigens that are immunodominant and optionally immunogenic when presented in vivo by MHC. Can be used. The MHC may be a class I or II MHC, and the peptide antigen may be a neoantige that may contain one or more amino acids essential for binding to the MHC. One or more amino acids of the neoantigen that are not essential for binding to MHC may contain one or more substitutions in the amino acid sequence compared to the wild-type sequence of the peptide. The peptide antigen may also be a self antigen that can stimulate T cells and induce an autoimmune response.

  [076] A self-antigen, a sequence complementary to the self-antigen, an antibody that specifically binds to the self-antigen, or a chimeric antigen receptor that specifically binds to the self-antigen reduces or prevents an autoimmune response It can be used for the manufacture of a medicament.

  [077] Peptide antigens may be cancer antigens that can stimulate T cells and induce an immune response, and may be included in a vaccine. A cancer antigen, a sequence complementary to the cancer antigen, an antibody that specifically binds to the cancer antigen, or a chimeric antigen receptor that specifically binds to the cancer antigen enhances or induces an immune response. Can be used for the manufacture of pharmaceuticals. T cells may be genetically modified and can include genetically modified T cell receptors that specifically bind to cancer antigens. The T cell may comprise a chimeric antigen receptor that specifically binds to a cancer antigen.

  [078] The peptide antigen may be a bacterial, viral, or microbial antigen. Viral antigens can stimulate T cells and induce an immune response. Viral antigens may be derived from cytomegalovirus (CMV) and / or function as a vaccine or part of a vaccine. A viral antigen, a sequence complementary to the viral antigen, an antibody that specifically binds to the viral antigen, or a chimeric antigen receptor that specifically binds to the viral antigen is used to produce a medicament that enhances or induces an immune response. Can be used for. T cells may be genetically modified and may include genetically modified T cell receptors that specifically bind to viral antigens. T cells may contain chimeric antigen receptors that specifically bind to viral antigens.

  [079] Bacterial antigens also stimulate T cells and induce an immune response so that they can function as a vaccine or part of a vaccine. A sequence complementary to the bacterial antigen, an antibody that specifically binds to the bacterial antigen, or a chimeric antigen receptor that specifically binds to the bacterial antigen is used for the manufacture of a medicament that enhances or induces an immune response May be. T cells may be genetically modified and may include genetically modified T cell receptors that specifically bind to bacterial antigens. T cells may contain chimeric antigen receptors that specifically bind to bacterial antigens.

[080] The composition disclosed above also comprises:
(A) contacting at least one T cell with the composition;
(B) upon activation, stimulated by at least one peptide of the composition comprising detecting at least one molecule secreted from at least one T cell, and (c) imaging at least one T cell May be used for identification of at least one T cell.

  [081] At least one T cell may be genetically modified, and at least one T cell may comprise a modified T cell receptor that specifically binds to at least one peptide of the composition. It may also include a chimeric antigen receptor (CAR) that specifically binds to at least one peptide of the composition. The imaging step may include contacting at least one T cell with one or more detectable agents that recognize one or more cell surface markers.

[082] Figure 1 shows a 9-mer peptide labeled with pCy5 antibody (W6 / 32), either with b2m subunit (upper plot) or without b2m subunit (lower plot, negative control) Number 7)-A pair of plots showing fluorescence intensity as a function of position for MHCI aggregates. Set 1 of Example 1 provides an experimental setup for the composition from which this data is derived. For further information on antibodies see Dao et al. (Science Translational Medicine March 13, 2013: Vol. 5, No. 176, p. 176ra33). [083] FIG. 2 is each 9 amino acids long, each having a unique sequence along the wild-type sequence of the WT1 protein, when bound to MHCI on the surface of the present disclosure and detected by a labeled antibody Based on its signal strength, or its predicted binding affinity as assessed by NetMHC 3.4, an algorithm widely used in the art to identify peptide antigens, It is a plot which shows WT1 peptide of. Any antibody that specifically recognizes the fully associated MHCI complex can be used to identify those peptides that will be presented in vivo by MHCI. This analysis was performed and the results compared to the predicted peptide identified to form a complex with MHCI by NetMHC3.4 and compared to the peptide predicted to form a complex with MHCI by NetMHC3.4. Of the 440 peptides plotted, NetMHC 3.4 identified 433 peptides as having too low affinity to bind to MHCI, in contrast, the theory that only 7 peptides bind to MHCI. Identified as having the ability to In marked contrast, the compositions and methods of the present disclosure have identified 18 peptides that actually bind to MHCI, including 13 peptides (top right quadrant) that the NetMHC3.4 algorithm will discard. . [084] Figure 3 is the same plot as shown in Figure 2, but with particular emphasis on the upper left quadrant. This quadrant is in accordance with the compositions and methods of the present disclosure that would be predicted by the algorithm to bind with sufficient affinity to MHCI when analyzed using the current industry standard method NetMHC to identify immunodominant peptides. Represents peptides with demonstrated binding ability to MHCI. Of particular interest is the highlighted peptide, referred to herein as WT1 peptide 126 (having the amino acid sequence RMFPNAPYL (SEQ ID NO: 7)). [085] Figure 4 is the same plot as shown in Figure 2, but with particular emphasis on the upper right quadrant. This quadrant is a composition of the present disclosure that will be discarded by the algorithm if it cannot be logically bound with sufficient affinity to MHCI when analyzed using the current industry standard method NetMHC to identify immunodominant peptides And peptides with demonstrated binding ability to MHCI according to methods. In other words, the compositions and methods of the present disclosure have empirically verified 13 peptides that would be false negatives when analyzed using only the NetMHC program. [086] Figure 5 is the same plot as shown in Figure 2, but with particular emphasis on the lower left quadrant. This quadrant is expected to bind with sufficient affinity to MHCI when analyzed using the current industry standard method NetMHC to identify immunodominant peptides, but is tested using the compositions and methods of the present disclosure Sometimes it represents a peptide that has been empirically shown not to form a fully associated peptide-MHCI complex. In other words, the compositions and methods of the present disclosure have empirically identified two peptides that would be false positives when analyzed using the NetMHC program only. [087] FIG. 6 is a plot showing the HLA-A2 loading specificity of the well-studied vaccinia virus peptide LMYDIINSV (SEQ ID NO: 8) associated with HLA-A2. The specificity of key residue interactions can be ascertained with substitution plots. [088] Substitution of each of the nine possible amino acids with each of the 20 possible amino acids to form an appropriate peptide-MHCI complex that is essential for the formation of the appropriate peptide-MHCI complex. The location was identified. The figure shows all 20 amino acids in one row, grouped by feature, with the amino acid single letter code: AFILMVWPGSYCQTNRKHDE. Amino acids A, F, I, L, M, V, W and P are nonpolar amino acids. Amino acids G, S, Y, C, Q, T, N are polar amino acids. Amino acids R, K and H are basic amino acids. Amino acids D and E are acidic amino acids. [089] FIG. 7 is a plot showing the ESK1 antibody binding specificity for peptide-MHC aggregates for the 9-mer peptide of WT1. The data shows the relative fluorescence intensity for a specific target (RMFPNAPYL (SEQ ID NO: 7)) and a cross-reactive target (RVPGVAPTL (SEQ ID NO: 9)). [090] FIG. 8 is a plot showing the ESK1 antibody binding specificity for the “RMFPNAPYL” (SEQ ID NO: 7) variant. Each amino acid in the 9 amino acid peptide was replaced with each of the 20 possible amino acids to identify the position of the amino acid within this peptide that is essential to form the appropriate peptide-MHCI complex. . The figure shows all 20 amino acids in one row, grouped by feature, with the amino acid single letter code: AFILMVWPGSYCQTNRKHDE. Amino acids A, F, I, L, M, V, W and P are nonpolar amino acids. Amino acids G, S, Y, C, Q, T, N are polar amino acids. Amino acids R, K and H are basic amino acids. Amino acids D and E are acidic amino acids. [091] FIG. 9 is a series of schematics demonstrating how a digital micromirror device (DMD) can be used to make the composition of the present disclosure by in situ synthesis of each peptide on the surface. FIG. The left panel shows an exemplary DMD that includes a plurality of micromirrors located directly on one surface of a series of three surfaces contained on a glass slide. Each of the plurality of micromirrors is independently programmed to tilt so that the light is shown on the plurality of micromirrors, and reflects light toward the surface area under the micromirror, or the micromirror Polarizes light from the surface area below. The tilt of the individual micromirrors is shown in the center panel. The right panel shows how a pulse of light controlled by the tilt of each micromirror builds the peptide on the surface in situ. The peptide of the composition of the present disclosure is attached to the surface by a linker at its C-terminus. Each amino acid added to the peptide contains a photolabile protecting group that prevents the addition of another amino acid to the peptide in the absence of light. However, when light contacts the protecting group, the amino acid is deprotected and an amino acid can be added. As amino acids flow across the surface, each micromirror reflects light at a pre-programmed time to deprotect the amino acid as the next amino acid intended in the peptide sequence flows across the surface. The area of the surface that is controlled by any given micromirror is referred to herein as a “microarea”. The microregion of the present disclosure has a virtual boundary, not a physical boundary. As shown in the right panel of this figure, in preferred embodiments of the surface of the present disclosure, there are no physical boundaries that interfere with the flow of amino acids across the surface during peptide synthesis. [092] FIG. 10A is a plot showing HLA-A2 / RLYDYFTRV peptide-MHC aggregate formation using a standard 5HEX linker (“RLYDYFTRV” is disclosed as SEQ ID NO: 10). Binding was performed overnight at 4 ° C. at pH 6.5. [093] FIG. 10B is a plot showing HLA-A2 / RLYDYFTRV peptide-MHC aggregate formation using a negatively charged HEX-asp-3HEX linker (“RLYDYFTRV” is disclosed as SEQ ID NO: 10) . Binding was performed overnight at 4 ° C. at pH 6.5. The dashed line shows an increase in the peptide-MHC association signal relative to the original peptide sequence when using a negatively charged linker as opposed to a standard 5HEX linker.

  [094] A T cell (also known as a thymocyte or T lymphocyte) is a type of lymphocyte (a type of white blood cell) that contains phagocytosis, antigen-specific cytotoxic T lymphocytes, and antigens. It plays a central role in cell-mediated immunity involving the release of various cytokines in response. Unlike antigen recognition by B cells, T cell recognition of antigens is not involved in direct binding to annoying antigens, but is a major histocompatibility complex that moves T cell receptors and pathogen-derived peptide epitopes and epitopes to the cell surface. It is involved in the interaction of body (MHC) molecules with complex surfaces. The human immune system is estimated to harbor approximately 25 million T cell clones with distinct specificities that define an extensive repository of evolving cellular immune responses against self and foreign antigens. Therefore, detecting and investigating these T cell populations is fundamentally and therapeutically important. Unfortunately, the extensive recognition ability of the human T cell repertoire is not well suited to current established methods for immune monitoring and T cell epitope discovery.

  [095] Methods often used to analyze antigen-specific T cell responses include intracellular cytokine staining, CD107 cytotoxicity assay, ELISpot, killing assay, and the like. These assays are all very useful in addressing certain T cell functions, but are often labor intensive, require large amounts of clinical peripheral blood mononuclear cell (PBMC) specimens, Poor resolution and / or low sensitivity to secretory response. Recently, staining of antigen-specific T cells with fluorescently labeled multimeric peptide-MHC complexes (p / MHC) has become widely used for the analysis of T cell responses to a small set of antigens. Yes. However, the synthesis of pMHC tetramers is time consuming and cannot be easily scaled. As a result, only a limited number of pMHC complexes can be investigated and it is therefore difficult to follow multiple T cell specificities for different functional events.

  [096] To overcome the limitations, the present disclosure provides for capturing and characterizing TCRs, TCR-like antibodies, and antigen-specific T cells based on their adhesion to p / MHC complexes. Provide an array-based approach.

  [097] Scaleable peptide microarrays are a paradigm shift in protein science. For example, thousands of targets can be tested simultaneously by synthesizing up to 2.9 million unique and spatially ordered peptides on a single surface using a digital micromirror device (DMD) can do. In addition, the compositions and methods of the present disclosure can incorporate modifications such as phosphorylation, unnatural amino acids such as citrulline, and restricted peptides (eg, cyclic peptides) into the peptide.

  [098] Another unique feature of the compositions of the present disclosure is the direct formation of p / MHC complexes at the surface. Traditionally, when p / MHC complexes are tested in a high-throughput format, each individual p / MHC complex is first constructed. Since the lack of MHC molecules (without the presence of the appropriate peptide) is unstable, both the peptide and the MHC component need to be present in the folding reaction. Multimers are then formed and spotted on the treated and / or derivatized surface. This manufacturing process can be a daunting task when multiple p / MHCs need to be tested. In addition, by spotting p / MHC on treated and / or derivatized surfaces, existing techniques suffer from effects that induce the surface to be inert, including protein denaturation and protein adsorption.

  [099] The methods of making the disclosed compositions introduce carrier molecules into the preparation process of MHC alpha and beta subunit mixtures that effectively rescue proteins from denaturation, even in the absence of suitable MHC binding peptides. By overcoming the technical hurdles of existing technology. The mixture is then applied directly to the array surface, where the presence of the bound peptide results in MHC refolding. Thus, thousands of pMHCs meet simultaneously. T cells, T cell receptors (TCR) (eg, natural and / or chimeric antigen receptors) or TCR-like antibodies can be applied to the surface. After incubation, peptide target specific T cells, for example, adhere to the corresponding p / MHC molecule, resulting in a spatial separation of different antigen specific T cell / TCR populations. Since assay readouts are position dependent rather than overall fluorescence signal, the disclosed compositions and methods can uniquely perform highly multiplex reactions.

In situ synthesis of peptides
[0100] In situ synthesis on the surface of a peptide or peptides of the present disclosure is performed quickly and efficiently using a patterning process. The process may be automated and computer controlled to allow creation of one or two dimensional arrays of peptides. A lithographic mask is not required, thus eliminating the significant costs and time delays associated with creating a lithographic mask, eliminating the need for time-consuming operations and alignment of multiple masks during the peptide array fabrication process.

  [0101] An active surface to which a peptide synthesis linker has been applied may be used to support the peptide being made. To provide a first level of amino acids starting from the active surface, a high-precision two-dimensional light image is projected onto the surface and the array on the active surface activated to bind to the first amino acid. Microregions (eg, also referred to as pixels or tiles in US Pat. No. 6,375,903 and US Pat. No. 8,030,477, which are hereby incorporated by reference in their entirety) are illuminated. Light incident on the microregion of the array to which the light is applied deprotects the bound amino acids and makes them available for further amino acid binding. After this development step, a fluid containing the appropriate amino acid is applied to the active surface and the selected amino acid binds to the exposed site. This process is then repeated until another amino acid is attached to a different set of microregion positions until all elements of the two-dimensional array on the surface have the appropriate amino acid attached to it (see, eg, FIG. 9). . The amino acids bound on the substrate are protected with a chemical that can bind to the amino acids, or a layer of photoresist that covers all of the bound amino acids, after which a new array pattern is projected and imaged onto the surface. Activating the protective material in those microregions where the first new amino acid is added. These microregions are then exposed and a solution containing the selected amino acids is applied to the array so that the amino acids bind to the exposed microregion locations. This process is then repeated for all other microregions of the second level amino acid. The described process can then be repeated for each desired level of amino acids until all of the selected two-dimensional array of peptide sequences is complete.

  [0102] Images to a micromirror device that includes a two-dimensional array of electronically addressable micromirrors, each of which can be selectively tilted between one of at least two separate positions. It is projected onto the surface using an image forming machine having a suitable light source that provides light. At one of the positions of each micromirror, light from an incident source on the micromirror is polarized off the optical axis and away from the surface, and at a second position of at least two positions of each micromirror, Are reflected toward the surface along the optical axis. The projection optical system receives light reflected from the micromirror and accurately forms the image on the active surface. Collimating optics can be used to collimate light from a light source into a beam that is provided directly to a micromirror array or beam splitter, where the beam splitter reflects a portion of the beam to the micromirror array. The light reflected from the micromirror array is sent through the beam splitter. Light reflected directly from the micromirror or transmitted through the beam splitter is directed to the projection optics lens, which images the micromirror array on the active surface. A selectively addressable micromirror in a micromirror array can fully reflect or fully polarize the light provided to it, so that the image of the micromirror array is between the “on” and “off” microregions. Shows very high contrast. Micromirrors can also be indexed to more than two positions, in which case additional optics are provided to allow exposure of more than one surface using a single micromirror array device. be able to. Furthermore, since the micromirror can reflect light of an arbitrary wavelength without damaging them, it is possible to use short-wavelength light including light in the range of ultraviolet to near ultraviolet from a light source.

  [0103] The micromirror array can be operated under computer control to provide an appropriate microregion address signal to the micromirror array to place the appropriate micromirror in its “reflective” or “polarized” position. The appropriate micromirror array pattern for each activation step at each level of amino acid added to the peptide is programmed into the computer controller. In this way, the computer control device controls the arrangement of images presented by the micromirror array in cooperation with the reagents provided on the surface.

  [0104] The surface may be transparent so that an image of the micromirror array can be projected through the surface. The surface may be mounted by enclosing and enclosing the active surface of the array within the flow cell, and through the flow cell, appropriate reagents can be flowed over the active surface of the array in the appropriate order to construct peptides into the array.

Major histocompatibility complex (MHC)
[0105] MHC class I or class II is expressed across the surface of all human nucleated cells. MHC class I (MHCI) complexes are present in all nucleated cells, whereas MHC class II (MHCII) complexes are present only in cells of the immune system (ie macrophages and lymphocytes). The MHC complex presents peptide fragments inside the cell, allowing the immune system to conduct body investigations for the presence of foreign invaders as determined by the presentation of non-self peptide sequences. In the case of an autoimmune condition, when the MHC presents a self peptide, the immune system is stimulated in a manner similar to that the immune system responds to non-self peptides. The leukocyte antigen genes that encode the components of MHC, including the human leukocyte antigen (HLA) gene, are very diverse and provide many possible permutations of both MHCI and MHCII MHC.

  [0106] Leukocyte antigen gene sequences, including human leukocyte antigen (HLA) gene sequences, are IPD-IMGT / HLA database (www.ebu.ac.uk/ipd/imgt/hla/) and UNIPROT (www.uniprot.org). Can be found in several publicly available databases including: The α chain of the MHCI complexes of the present disclosure may be modified to remove transmembrane, hinge and / or cytoplasmic regions. To accomplish this modification, the full-length nucleic acid sequence of the MHCI complex alpha chain is obtained from a public database, edited to remove sequences encoding transmembrane, hinge and / or cytoplasmic regions, and optionally Back-translate using a codon table optimized for host T cells (eg, E. coli) in which the nucleic acid is expressed. The α chain of the MHCII complex of the present disclosure may be modified to remove transmembrane and / or cytoplasmic regions. To accomplish this modification, the full-length nucleic acid sequence of the MHCII complex α-chain is obtained from a public database and edited to remove sequences encoding transmembrane and / or cytoplasmic regions, and optionally nucleic acids. Is back-translated using a codon table optimized for host T cells (eg, E. coli) in which is expressed. The β chain of the MHCII complex of the present disclosure may be modified to remove transmembrane and / or cytoplasmic regions. To accomplish this modification, the full length nucleic acid sequence of the β chain of the MHCII complex is obtained from a public database, edited to remove sequences encoding transmembrane and / or cytoplasmic regions, and optionally nucleic acid Is back-translated using a codon table optimized for host T cells (eg, E. coli) in which is expressed.

  [0107] Peptides and MHC components associate with each other in the endoplasmic reticulum (ER) of each cell before being presented on the cell surface. In vivo, peptides that form a complex with MHC are a byproduct of the degradation of cytoplasmic proteins by the proteasome. The peptides of the present disclosure can comprise or consist of any sequence, and the sequence can be derived from any polypeptide. For example, a peptide of the present disclosure may contain one amino acid (or 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) at each step from the N-terminus to the C-terminus of the polypeptide sequence, eg Designed by moving systematically at 6-30 amino acid intervals (ie, the length of the peptide intended to be synthesized on the surface of the disclosed composition) until the length of the polypeptide is traversed can do. In addition, using the set of peptide sequences generated by moving along the sequence in the process, additional sets of peptides can be used, for example, the amino acids present in each one sequence of those peptides. Can be expanded by substituting each one of the amino acids to provide all possible sequence variations. In this example, as in Example 1, when the WT1 protein is divided into peptides having 9 amino acids, one amino acid is transferred at each step, the set of peptides provided in FIG. 1 is generated. It will be. Subsequently, if these peptides are substituted as shown in Example 1, a further set of peptides, ie a replacement set, can be generated. This process can be applied to any polypeptide to produce at least one peptide or peptides of the composition of the present disclosure.

  [0108] Challenges to identify intervention points that inhibit or stimulate the immune system define rules that allow some peptides to be loaded into the MHC while others are not loaded It is to be. Using a highly multiplexed format, the compositions and methods of the present disclosure provide empirical evidence showing a demonstrated ability or inability to associate with MHCI and / or MHCII of peptides.

  [0109] When a peptide is presented on the surface of a cell as part of a stable MHC complex, the immune system (eg, T cells) can use the peptide-MHC complex to find sequences that are foreign or non-self antigens. Sampling the body. Under autoimmune conditions, the immune system cannot distinguish between self-antigens and non-self-antigens, ultimately treating cells that exhibit self-antigens as foreign invaders and attacking healthy tissue. In the same highly multiplexed reaction used to identify immunodominant peptides, the compositions and methods of the present disclosure make the subject immune system against cancer cells or the immune system is better with infection. Can be used to identify peptides that stimulate the immune system, including synthetic peptides, modified peptides and / or neoantigens that can be used to stimulate to fight. The compositions and methods of the present disclosure provide for the ability of T cells to contain peptide-MHC complexes on cells that are not recognized by the natural immune system, eg, chimeric antigen receptors that identify peptide-MHC complexes on cancer cells. Can be used to verify.

Definition
[0110] As used in this disclosure, the singular forms "a", "and", and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods, a reference to “a dose” includes one or more doses and equivalents known to those of skill in the art, and the like.

  [0111] The term "about" or "approximately" means within an acceptable error range for a particular value determined by one of ordinary skill in the art, and is a method of measuring or determining a value, eg, measurement Partly depends on system limits. For example, “about” can mean within one or more standard deviations. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can preferably mean within 5 times, more preferably within 2 times the value. Unless stated otherwise, when a particular value is described in this application and in the claims, the term “about” is presumed to mean within an acceptable error range for the particular value. .

  [0112] The present disclosure provides isolated or substantially purified polynucleotide or protein compositions. An “isolated” or “purified” polynucleotide or protein or biologically active portion thereof is substantially a component that normally accompanies or interacts with a polynucleotide or protein found in its natural environment. Or essentially not. Thus, an isolated or purified polynucleotide or protein, when produced by recombinant techniques, is substantially free of other cellular material or culture medium and is chemically synthesized when chemically synthesized. It is essentially free of body or other chemicals. Optimally, an “isolated” polynucleotide comprises a sequence that is naturally contiguous in the polynucleotide in the genomic DNA of the organism from which the polynucleotide is derived (ie, sequences located at the 5 ′ and 3 ′ ends of the polynucleotide). Not included (optimally protein coding sequence). For example, in various embodiments, the isolated polynucleotide has a nucleotide sequence that naturally flank the polynucleotide of the genomic DNA of the cell from which the polynucleotide is derived from about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb. Or less than 0.1 kb. Proteins substantially free of cellular material include protein preparations in which contaminating protein is less than about 30%, 20%, 10%, 5%, or 1% (by dry weight). When the protein of the invention, or biologically active portion thereof, is produced recombinantly, an optimal culture medium is about 30% (by dry weight) containing a chemical precursor or a chemical other than the protein of interest. 20%, 10%, 5% or less than 1%.

  [0113] The present disclosure provides the disclosed DNA sequences and protein fragments and variants encoded by these DNA sequences. As used throughout this disclosure, the term “fragment” refers to a portion of a DNA sequence, or a portion of an amino acid sequence and thus the protein encoded thereby. A fragment of a DNA sequence that includes a coding sequence can encode a protein fragment that retains the biological activity of the native protein, and thus DNA recognition or binding activity to the target DNA sequence described herein. Alternatively, fragments of DNA sequences that are useful as hybridization probes generally do not encode proteins that retain biological activity or retain promoter activity. Thus, a fragment of a DNA sequence can range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides up to the full length polynucleotide of the invention.

  [0114] Nucleic acids or proteins of the present disclosure can be constructed by a modular approach involving pre-assembled monomer units and / or repeat units in a target vector that can then be assembled into a final target vector. The polypeptides of the present disclosure may comprise the repetitive monomers of the present disclosure and can be constructed by a modular approach with temporary assembly repeat units in a target vector that can be subsequently assembled into a final target vector. The present disclosure provides polypeptides made by the present methods, as well as nucleic acid sequences that encode these polypeptides. The present disclosure provides host organisms and cells that contain nucleic acid sequences that encode a polypeptide made by this modular approach.

  [0115] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide or protein. . If the polynucleotide is derived from genomic DNA, expression can include splicing of mRNA in eukaryotic cells.

  [0116] "Binding" refers to a sequence specific non-covalent interaction between macromolecules (eg, between a protein and a nucleic acid). As long as the interaction is sequence specific as a whole, not all components of the binding interaction need be sequence specific (eg, contact with phosphate residues in the DNA backbone).

  [0117] A "binding protein" is a protein that can bind non-covalently to another molecule. The binding protein can bind to, for example, a DNA molecule (DNA binding protein), an RNA molecule (RNA binding protein) and / or a protein molecule (protein binding protein). In the case of a protein binding protein, the protein can bind to itself (and form homodimers, homotrimers, etc.) and / or one of the different protein (s). Or it can bind to multiple molecules. A binding protein can have more than one type of binding activity. For example, zinc finger proteins have DNA binding, RNA binding and protein binding activities.

  [0118] The term "comprising" is intended to mean that the compositions and methods include, but do not exclude, the listed elements. `` Consisting essentially of '' when used to define compositions and methods, excludes other elements that have any significant significance to the combination when used for the intended purpose. means. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants or inert carriers. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition phrases are within the scope of this invention.

  [0119] "Linked" or "operably linked" or equivalents thereof (eg, "operably linked") are those where two or more molecules are one or both molecules or It means that they are arranged with each other so that they can interact to affect the function resulting from the combination. A peptide and its corresponding linker can be operably linked.

  [0120] The peptides of the present disclosure can comprise an epitope of an antibody used to detect a peptide or peptide-MHC complex. Further, the antibody can be contacted with a composition of the present disclosure to determine the epitope of the antibody, including naturally occurring antibodies. The term “epitope” refers to an antigenic determinant of a polypeptide. An epitope can contain three amino acids in a spatial conformation unique to the epitope. In general, an epitope consists of at least 4, 5, 6 or 7 such amino acids, and more usually consists of at least 8, 9, or 10 such amino acids. Methods for determining the spatial conformation of amino acids are known in the art and include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance.

  [0121] The term "antibody" is used in the broadest sense and specifically encompasses single monoclonal antibodies (including agonist and antagonist antibodies) as well as antibody compositions having polyepitope specificity. It is within the scope to use natural or synthetic analogs, variants, variants, alleles, homologs and orthologs (collectively referred to herein as “analogs”) of the antibody as defined herein. It is. Thus, according to one embodiment thereof, the term “the antibody” in its broadest sense also encompasses such analogs. In general, in such analogs, one or more amino acid residues may be substituted, deleted and / or added as compared to an antibody as defined herein.

  [0122] Antibodies used to detect the peptides and / or peptide-MHC complexes of the present disclosure may be raised from any species. In certain embodiments, these antibodies have human CDR sequences, even if the framework regions are from a non-human species. In certain embodiments, these antibodies are fully human, but may include one or more modifications to be non-natural. In certain embodiments, these antibodies are fully in vitro to mimic the ability of the human immune system to recognize the disclosed peptides and / or peptide-MHC complexes bound to the surface of the disclosed compositions. A human being.

[0123] Antibody fragments may be incorporated into a detectable agent for recognizing the peptides and / or peptide-MHC complexes of the present disclosure bound to the surface of the compositions of the present disclosure. “Antibody fragments” and grammatical variations thereof, as used herein, are defined as part of an intact antibody comprising the antigen binding site or variable region of the intact antibody, wherein part of the intact antibody It does not include the constant heavy chain domain of the Fc region (ie, CH2, CH3, and CH4 depending on the antibody isotype). Examples of antibody fragments include Fab, Fab ′, Fab′-SH, F (ab ′) ₂ , and Fv fragments; diabodies; polypeptides having a primary structure consisting of one uninterrupted sequence of consecutive amino acid residues Any antibody fragment (referred to herein as a “single chain antibody fragment” or “single chain polypeptide”), such as, but not limited to, (1) a single chain Fv (scFv) molecule, (2) a single chain polypeptide having only one light chain variable domain, or a fragment thereof containing three CDRs of the light chain variable domain without an associated heavy chain portion, and (3) one heavy chain variable region Single-chain polypeptide containing only, or a fragment containing the three CDRs of the heavy chain variable domain without an associated light chain portion; and multispecific or multivalent structures formed from antibody fragments It is included. For antibody fragments comprising one or more heavy chains, the heavy chain can comprise any constant domain sequence found in the non-Fc region of the intact antibody (eg, CHI of the IgG isotype) and / or intact antibody And / or a leucine zipper sequence or constant domain sequence fused or placed in the heavy chain hinge region sequence. The term further includes a single domain antibody ("sdAB") that generally refers to an antibody fragment having a single monomer variable antibody domain (eg, from a camelid). Such antibody fragment types are readily understood by those skilled in the art.

  [0124] The term "scFv" refers to a single chain variable fragment. scFv is an immunoglobulin heavy chain (VH) and light chain (VL) variable region fusion protein connected by a linker peptide. The linker peptide can be about 5-40 amino acids in length, or about 10-30 amino acids, or about 5, 10, 15, 20, 25, 30, 35, or 40 amino acids. Single chain variable fragments lack the constant Fc region found in intact antibody molecules, and thus the common binding site (eg, protein G) used to purify antibodies. The term further includes intrabodies, scFvs, antibodies that are stable in the cytoplasm of a cell and can bind to intracellular proteins.

  [0125] The term "single domain antibody" refers to an antibody fragment having a single monomeric variable antibody domain that can selectively bind to a particular antigen. Single domain antibodies are generally about 110 amino acid long peptide chains, including heavy chain antibody variable domains (VH) or general IgG variable domains, generally against the same antigen as the whole antibody. Has affinity but is more heat resistant and more stable to surfactants and high concentrations of urea. Examples are antibodies derived from camelid or fish antibodies. Alternatively, single domain antibodies can be made from common mouse or human IgG having four chains.

  [0126] As used herein, the terms "specifically binds" and "specific binding" refer to antibodies, antibody fragments that preferentially bind to a particular antigen present in a homogeneous mixture of different antigens. Or refers to the ability of Nanobody. In certain embodiments, the specific binding action is greater than about 10 times or higher (eg, about 1000 times or higher) between desirable and undesired antigens in a sample, in some embodiments. (Over 10,000 times). “Specificity” refers to the ability of an immunoglobulin fragment, such as an immunoglobulin or Nanobody, to bind preferentially to one antigenic target for a variety of antigen labels and does not necessarily imply high affinity.

  [0127] The term "nucleic acid" or "oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked together. The single strand description also defines the sequence of the complementary strand. Thus, the nucleic acid can include the single-stranded complementary strand shown. Nucleic acids of the present disclosure also include substantially identical nucleic acids that retain the same structure or encode the same protein, or complements thereof.

  [0128] The nucleic acids of the present disclosure may be single-stranded or double-stranded. The nucleic acids of the present disclosure can include double stranded sequences even though the majority of the molecules are single stranded. The nucleic acids of the present disclosure can include a single stranded sequence even though the majority of the molecule is double stranded. The nucleic acids of the present disclosure can include genomic DNA, cDNA, RNA, or hybrids thereof. The nucleic acids of the present disclosure can include deoxyribo- and ribo-nucleotides. The nucleic acids of the present disclosure can comprise a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine. The nucleic acids of the present disclosure can be synthesized to include unnatural amino acid modifications. The nucleic acids of the present disclosure can be obtained by chemical synthesis methods or recombinant methods.

  [0129] The nucleic acids of the present disclosure may be non-naturally occurring in their entire sequence or any portion thereof. The nucleic acids of the present disclosure may include one or more non-naturally occurring mutations, substitutions, deletions or insertions that render the entire nucleic acid sequence non-natural. The nucleic acids of the present disclosure can include one or more overlapping, inverted or repetitive sequences in which the resulting sequence is non-naturally occurring and the resulting sequence is non-naturally occurring. The nucleic acids of the present disclosure can include non-naturally occurring modifications, artificial or synthetic nucleotides that render the entire nucleic acid sequence non-natural.

  [0130] Given the degeneracy in the genetic code, multiple nucleotide sequences can encode any particular protein. All such nucleotide sequences are contemplated herein.

  [0131] As used throughout this disclosure, the term "variant" used to describe a nucleic acid is (i) a portion or fragment of a reference nucleotide sequence, (ii) the complement of a reference nucleotide sequence or portion thereof. (Iii) a nucleic acid that is substantially identical to a reference nucleic acid or its complement, or (iv) a nucleic acid that hybridizes under stringent conditions to a reference nucleic acid, its complement or a sequence substantially identical thereto. .

  [0132] As used throughout this disclosure, the term "vector" refers to a nucleic acid sequence that includes an origin of replication. The vector can be a DNA or RNA vector. The vector may be a self-replicating extrachromosomal vector, preferably a DNA plasmid.

  [0133] As used throughout this disclosure, the term "variant" when used to describe a peptide or polypeptide differs in amino acid sequence by amino acid insertions, deletions or conservative substitutions, Refers to a peptide or polypeptide that retains at least one biological activity. Furthermore, a variant can also mean a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity.

  [0134] Conservative substitutions of amino acids, ie replacing amino acids with different amino acids with similar properties (eg, hydrophilicity, charged region extent and distribution) are typically recognized as being accompanied by minor changes . As understood in the art, these minor changes can be identified in part by considering the amino acid hydropathy index. Kyte et al. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on consideration of its hydrophobicity and charge. Amino acids with similar hydropathic indices can still retain protein function upon substitution. In one aspect, amino acids having a hydropathy index of ± 2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that result in proteins that retain biological function. Considering the hydrophilicity of amino acids in the peptide context allows for the calculation of the peptide's maximum local average affinity, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101, incorporated herein by reference.

  [0135] Substitution of amino acids with similar hydrophilicity values can result in peptides that retain biological activity, eg, immunogenicity. Substitutions can be made with amino acids having hydrophilicity values within ± 2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by the specific side chain of the amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function depend on the relative similarity revealed by the hydrophobicity, hydrophilicity, charge, size and other properties of amino acids, especially amino acid side chains Then it is understood.

  [0136] As used herein, "conservative" amino acid substitutions may be defined as shown in Tables A, B, or C below. In some embodiments, the fusion polypeptide and / or nucleic acid encoding such a fusion polypeptide comprises conservative substitutions introduced by modification of a polynucleotide encoding a polypeptide of the invention. Amino acids can be classified according to physical properties and contributions to secondary and tertiary protein structure. A conservative substitution is the replacement of one amino acid with another having similar properties. Exemplary conservative substitutions are shown in Table A.

[0137]

  [0138] Alternatively, conservative amino acids can be found in Lehninger (Biochemistry, 2nd edition; Worth Publishers, Inc., NY, NY (1975), pages 71-77) as shown in Table B. Can be classified as described.

[0139]

  [0140] Alternatively, exemplary conservative substitutions are shown in Table C.

[0141]

  [0142] Polypeptides of the present disclosure include polymorphs that retain one or more insertions, deletions or substitutions of amino acid residues, or any combination thereof, and modifications other than insertions, deletions or substitutions of amino acid residues. It should be understood that it is intended to include peptides. A polypeptide or nucleic acid of the present disclosure can include one or more conservative substitutions.

  [0143] As used throughout this disclosure, the term "more than one" of the foregoing amino acid substitutions is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20 or more referenced amino acid substitutions. The term “more than one” can refer to more than 2, 3, 4 or 5 referenced amino acid substitutions.

  [0144] Polypeptides and proteins of this disclosure may be those whose entire sequence or any portion thereof does not exist in nature. The polypeptides and proteins of the present disclosure may include one or more non-naturally occurring mutations, substitutions, deletions or insertions that render the entire amino acid sequence non-natural. The polypeptides and proteins of the present disclosure may include one or more overlapping, inverted or repetitive sequences that make the entire amino acid sequence non-naturally occurring and the resulting sequence does not naturally occur. The polypeptides and proteins of the present disclosure may include non-naturally occurring modifications, artificial or synthetic amino acids that render the entire amino acid sequence non-natural.

  [0145] As used throughout this disclosure, "sequence identity" is for blasting two sequences that can be searched using the default parameters from the National Center for Biotechnology Information (NCBI) ftp site. Can be determined using the stand-alone executable BLAST engine program (bl2seq) of Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250, which is incorporated herein by reference in its entirety. ). As used in the context of two or more nucleic acid or polypeptide sequences, “identical” or “identity” refers to a specified percentage of residues that are identical over each specified region of the sequence. The percentage aligns the two sequences optimally, compares the two sequences over a particular region, determines the number of positions where identical residues exist in both sequences, and obtains the number of matching positions This can be calculated by dividing the number of positions performed by the total number of positions in a particular region and multiplying the result by 100 to obtain the percentage sequence identity. If the two sequences are of different lengths or the alignment results in one or more staggered ends and the comparison contains only a single sequence in a particular region of comparison, then the residue of the single sequence is It is included in the denominator of the calculation but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity may be performed manually or using a computer sequence algorithm such as BLAST or BLAST 2.0.

  [0146] Unless otherwise noted, all percentages and ratios are calculated on a weight basis.

  [0147] Unless otherwise noted, all percentages and ratios are calculated based on the total composition.

  [0148] Any maximum numerical limitation set forth throughout this disclosure includes such low numerical limits as if any low numerical limitation was expressly set forth herein. Any given minimum numerical limitation set forth throughout this disclosure includes all higher numerical limitations as if such large numerical limitations were expressly set forth herein. Any given numerical range set forth throughout this disclosure may be used to express any narrow numerical range that falls within such wide numerical range, as if all such narrow numerical ranges were expressly set forth herein. Included.

  [0149] The values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such value is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a value disclosed as “20 μm” is intended to mean “about 20 μm”.

Example 1: HLA Class I prepared and association human leukocyte antigen (HLA) on the array and beta _{2-microglobulin} (b2m) Origin of protein:
[0150] Cloning and expression in E. coli
[0151] HLA-A ^* 02 ^: 01 and b2m were provided by Roche Glycart AG (Schlieren, Switzerland) and Roche Diagnostics GmbH (Penzberg, Germany).

[0152] HLA-A ^* 11: 01, HLA-B ^* 07: 02 and HLA-C ^* 07 ^: 02 amino acid sequences from the UniProt database were cleaved to produce hinge, transmembrane and cytoplasmic regions at the C-terminus, N The leader peptide sequence was removed from the end.

[0153] HLA-A * 11: 01:
MGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGYYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO: 1)

[0154] HLA-B * 07: 02:
GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQIYKAQAQTDRESLRNLRGYYNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQYAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQRRAYLEGECVEWLRRYLENGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO: 2)

[0155] HLA-C * 07: 02
SHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQADRVSLRNLRGYYNQSEDGSHTLQRMSGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADTAAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWE (SEQ ID NO: 3)

[0156] The amino acid sequences for HLA-A ^* 11: 01, HLA-B ^* 07: 02 and HLA-C ^* 07 ^: 02 proteins were back-translated into DNA sequences using the E. coli optimal codon table and It was synthesized as a strand DNA and cloned into plasmid DNA by DNA 2.0 (www.dna20.com). The protein was expressed in E. coli in the form of inclusion bodies by either DNA 2.0 (HLA-A ^* 11 ^: 01) or Penzberg (HLA-B ^* 07 ^: 02 and HLA-C ^* 07 ^: 02). Inclusion bodies with expressed protein were stored at −80 ° C. prior to solubilization.

[0157] Solubilization of inclusion bodies
[0158] 100 mg of inclusion bodies were transferred to a 2.0 ml Eppendorf tube, resuspended in 1 ml of pH 7.8 50 mM Tris-HCl, 5 mM EDTA, 1% Tween 20, 2 mM DTT, 2,000 Centrifuged at xg for 2 minutes. The supernatant was removed and the pellet was loosened by vortexing.

  [0159] The pellet was mixed with 1 ml of 25 mM Tris-HCl, pH 7.8, 2 M NaCl, 2 M urea, 2 mM DTT and centrifuged at 4,000 × g for 2 min. The supernatant was removed and the pellet was loosened by vortexing.

  [0160] The pellet was mixed with 1 ml of 1 × PBS, 0.5 mM PMSF and centrifuged at 2,000 × g for 2 minutes. The supernatant was removed and the pellet was loosened by vortexing.

  [0161] The pellet was dissolved in 1 ml of 20 mM Tris-HCl, pH 7.8, 8 M urea, 100 μM β-mercaptoethanol and stored overnight at 4 ° C. for complete dissolution. The protein concentration was determined assuming an absorbance of 280 nm and one A280 unit = 1 mg / ml. Protein purity was stained with EZblue staining reagent (Sigma) and tested using 12% NuPAGE Bis-Tris gel and MES running buffer (Novex).

Preparation of HLA class I complexes and association on arrays
[0162] Preparation of HLA / b2m complex
[0163] Typically, 630 μl of 10 mM Tris-HCl pH 8.5, 20 μl of 10% BSA in 20 mM Tris-HCl pH 7.8, 300 μg of 30 mg / ml solubilized b2m and 600 μg of 15-40 mg / Mix with ml solubilized alpha chain in the order shown at room temperature (RT). The control sample did not contain b2m protein. The mixed sample was incubated overnight at 4 ° C. and centrifuged at 12,000 × g for 4 minutes to remove the precipitate. The supernatant was concentrated with an Amicon Ultra 10K filter (Millipore) using approximately 400 μl of two sample loads and centrifuged at 12,000 × g for 2 minutes each. The sample buffer was replaced with 10 mM Tris pH 8.5 by adding 350 μl 10 mM Tris-HCl pH 8.8 to the filter holding volume and concentrated by centrifugation at 12,000 × g for 4 minutes. The buffer exchange procedure was repeated two more times, using 350 μl of 10 mM Tris-HCl, pH 8.5 each time. After the final centrifugation step, the retention volume (approximately 100 μl) is collected into a fresh tube by centrifugation at 1,000 × g for 2 minutes, filtered through a 5.0 μm Ultrafree filter (Millipore) and stored at 4 ° C. did.

[0164] Complex Association and Detection on HLA Class I Arrays
[0165] Peptide array slides after synthesis and deprotection were stored in sealed containers at -20 ° C or used immediately. Prior to sample application, slides were treated with 0.7 μg / ml Cy-5 labeled streptavidin (Amersham) 1 × binding buffer (1% casein, 10 mM Tris pH 7.4) for array blocking and fiducial mark staining. , 0.25% Tween 20) for 1 hour at room temperature, rinsed with water and dried by centrifugation for 30 seconds using a tabletop centrifuge equipped with a slide holder.

  [0166] The prepared HLA class I samples were loaded into an incubation chamber attached to the surface of the peptide array without any additional treatment or dilution. After overnight incubation at room temperature, the incubation chamber is removed, the array is rinsed with water, and the HLA complexes are washed at room temperature with anti-HLA-A, B, C conformation antibodies (Alexa647-MEM123 (Novus) 1 × binding buffer). (300-fold dilution) for 1 hour. After staining, the array was rinsed with water, dried and scanned at 635 nm.

Parameters affecting peptide / HLA complex association on the array:
[0167] Influential parameters for successful peptide / HLA / b2m association:
[0168] For this example, the optimal concentration of BSA was found to be 2-3% added to the HLA / b2m mixture. Without BSA, complex formation was not observed.

  [0169] The optimal HLA for the b2 mmol / molar ratio for the complex preparation for this example was found to be 1: 1 to 1: 2.

[0170] In the process of HLA / b2m mixture preparation, HLA-B ^* 07 ^: 02 and HLA-C ^* 07 ^: 02 proteins formed precipitates while diluted from 8M urea stock. This effect is pH dependent and can be minimized using 10 mM pH 8.5 Tris-HCl buffer.

  [0171] The complex association intensity signal is proportional to the HLA / b2m concentration. For optimal concentration, two factors: first, increasing HLA / b2m concentration increases both specific and non-specific signal (background), second, HLA allele has HLA / b2m concentration It should be taken into account that it varies depending on the signal for. This means that the optimal concentration should be found independently for each allele. The conditions described in the “HLA / b2m complex preparation” section can be used as a template for optimization.

  [0172] At least overnight incubation is recommended for both HLA / b2m complex preparation and peptide / HLA / b2m complex association on the array.

  [0173] In order to optimize the linker for peptide binding to the slide surface, a 3: 1 mix of 3 different linkers, hexanoic acid, PEG and G / S amino acids, 5 different 1-5 Tested in length. A linker consisting of three hexanoic acid molecules has been found to be optimal using the signal / background ratio as a metric. Furthermore, the linker can be optimized to contain at least one negative monomer, especially when the surface has a net positive charge. For example, the linker may comprise at least one negatively charged amino acid.

  [0174] Cy5-labeled W6 / 32 (NBP2-00439) or Alexa647-labeled MEM123 (NB500-505AF647) (both from Novus Biological), conformational antibodies that detect the HLA / b2m complex, were assembled on the peptide array. Can be used at optimal antibody dilution factors of 1: 100 and 1: 300, respectively, for detection of HLA / b2m.

[0175] Parameters with minimal or negative effect on peptide / HLA / b2m association:
[0176] Several reagents have been reported in the literature as important for efficient peptide / HLA / b2m complex association. Among them, 0.4 M L-arginine, 0.25% glucopyranoside, 5 mM reduced L-glutathione / 0.5 mM oxidized L-glutathione, L-GL dipeptide, etc. Are auxiliary peptides and low-affinity peptides. These reagents were found to have minimal or inhibitory effects on HLA / b2m complex association on peptide arrays.

[0177] Other parameters including pH ranging from 5.5 to 8.5, 20 mM to 1 M NaCl or KCl, 1 mM MgCl ₂ or CaCl ₂ , 0.1% casein, 60 mM urea and peptides It was found to have minimal or inhibitory effects on HLA / b2m complex association on the array.

  [0178] Several other antibodies were tested for detection of associated HLA / b2m complexes: anti-b2m, anti-HLA MEM81, MEM147 and BB7.2. These antibodies all showed low signal or high background compared to w6 / 32 and MEM123 antibodies, except for BB7.2, which showed a good signal / background ratio. However, BB7.2 was not used in this study due to limitations in HLA-A and the inability to detect HLA-B and HLA-C alleles.

Example 2: MHCI surface array design set 1
[0179] 12plex layout:
[0180] Batch treated 9-mer peptide (123,675 peptide): 5 control proteins; NY-ES01, WT1, MAGE3, MAGE4, FOXP3; 3 different linker types (PEG8, 6-aminohexanoic acid, Gly: SER 4: 1 mix) and 1 amino acid step size tiled 9-mer peptide representing 5 different linker lengths.

[0181]

  [0182] Initial experimental designs to optimize pMHC formation and detection consisted of 5 known cancer proteins, 3 different peptide linker types, and 5 different linker lengths (1-5 repeats / monomer ) Included.

Set 2
[0183] 12-plex layout: two peptides from Wilms tumor and one other peptide. Wilms tumor is a rare kidney cancer also known as nephroblastoma.

[0184] Single amino acid substitution analysis (40,500 peptides): Three well-studied control peptides are synthesized where the corresponding antibodies are available and can recognize peptides bound to MHCI. The antibody has nanomolar binding affinity and, therefore, the antibody is expected to specifically bind to the peptide-MHCI complex. Twenty peptides are synthesized by substituting a total of 20 amino acids for each position of the 9-mer peptide. In the 9mer, 20 ^* 9 = 180 peptides are required to perform a complete single amino acid substitution analysis. Three different peptide linker types (PEG8, 6-aminohexanoic acid, Gly: SER 4: 1 mix) and five different linker lengths (linker length is the number of synthesis cycles for each linker monomer ½ Defined as / 3/4/5). All peptides are tested in 5 replicates with each linker type and length.

  Variables under set 1 and 2 optimization

[0185]

  [0186] For both sets 1 and 2, the pMHC complex was successfully formed on the surface.

  [0187] For set 1, antibody W6 / 32 (Novus Biosciences) labeled with pCy5 was added to the surface and allowed to bind to any appropriately associated pMHC complex. In the upper panel of FIG. 1, each peak is a separate pMHC complex. Some complexes have been reported in the literature, while other complexes have not been reported. Most complexes have a leucine at the second position (positions 2 and 8 are considered anchor positions). FIG. 1 (bottom panel) is a negative control with only HLA α subunit (“b2m” or no β subunit).

Example 3: Identification of immunodominant peptides and comparison with NetMHC3.4
[0188] The compositions and methods of the present disclosure can be used to identify those peptides that would be immunodominant when presented in vivo by MHC. Since the composition of the present disclosure can contain at least 10 ⁶ unique peptides on the surface simultaneously, any antigen of interest, for example, all 9 amino acid sequences linked to the MHCI complex on the surface Can be presented in permutation. In a single highly multiplexed experiment, any antibody that specifically recognizes a fully and properly associated peptide-MHCI complex is used to easily identify peptides that can be presented in vivo. In the same experiment, introducing one or more T cells to the surface to determine which of the peptides contained in the appropriately associated peptide-MHCI complex stimulates one or more T cells. it can. These two criteria, both of which form the appropriate peptide-MHC complex and stimulate T cells, can be determined in a single experiment for at least 10 ⁶ unique peptides. ) Only, the compositions and methods of the present disclosure provide an excellent means for in vitro identification of those peptides that would be immunodominant when presented in vivo by MHC.

  [0189] The ability of the disclosed compositions and methods was compared to the ability of the computer algorithm NetMHC3.4, the current industry standard method for predicting immunodominant peptides.

  [0190] Figure 2 is each 9 amino acids in length, each having a unique sequence along the wild type sequence of the WT1 protein, when bound to MHCI on the surface of the present disclosure and detected by a labeled antibody Provides a plot showing multiple WT1 peptides organized in quadrants based on their signal intensity, or their predicted binding affinity as assessed by NetMHC3.4. Any antibody that specifically recognizes the fully associated MHCI complex can be used to identify those peptides that will be presented in vivo by MHCI.

  [0191] Of the 440 peptides plotted, NetMHC 3.4 identified 433 peptides as having too low affinity to bind to MHCI, in contrast, only 7 peptides to MHCI Identified as having a theoretical ability to bind. In marked contrast, the compositions and methods of the present disclosure have identified 18 peptides that actually bind to MHCI, including 13 peptides (upper right quadrant) that the NetMHC3.4 algorithm will discard. .

  [0192] Figure 3 highlights the upper left quadrant. The upper left quadrant represents the compositions and methods of the present disclosure that would be predicted by the algorithm to bind with sufficient affinity to MHCI when analyzed using the current industry standard method NetMHC to identify immunodominant peptides. Thus, it represents a peptide with demonstrated ability to bind to MHCI. Of particular interest is the highlighted peptide, referred to herein as WT1 peptide 126.

  [0193] FIG. 4 highlights the upper right quadrant. The upper right quadrant is the composition of the present disclosure that would be discarded by the algorithm if it could not logically bind to MHCI with sufficient affinity when analyzed using the current industry standard method NetMHC to identify immunodominant peptides. Represents peptides having the ability to bind to MHCI demonstrated according to the methods and methods. In other words, the compositions and methods of the present disclosure have empirically verified 13 peptides that would be false negatives when analyzed using only the NetMHC program.

  [0194] Figure 5 highlights the lower left quadrant. The lower left quadrant would be expected to bind with sufficient affinity to MHCI when analyzed using the current industry standard method NetMHC to identify immunodominant peptides, but using the compositions and methods of the present disclosure Represents a peptide empirically shown to not form a fully associated peptide-MHCI complex. In other words, the compositions and methods of the present disclosure have empirically identified two peptides that would be false positives when analyzed using the NetMHC program only.

  [0195] The MHC components are diverse, and there are almost infinite possible sequences for peptides that form complexes with MHC, so the ER environment can be reproduced, and the vastness of peptides that can be used with any component of MHC Advanced empirically to determine which of the peptide-MHC complexes can subsequently stimulate the immune system (eg, T cells) There is a long-needed and unmet need for systems that are multiplexed into the system.

  [0196] Computer modeling is inadequate for this complex system. The reason is that, as shown in this disclosure, many peptide-MHC complexes that are actually formed are not expected to associate using the existing algorithms. Conversely, some of the peptide-MHC complexes that are expected to associate have been shown to form unstable complexes by the tests presented in this disclosure.

  [0197] To further demonstrate the capabilities of the highly multiplexed experience-based method of the present disclosure, FIGS. 7 and 8 show that “RMFPNAPYL” (SEQ ID NO: 4) that ESK1 can bind with varying affinities. 7) Provide variant sequence analysis. Since the array of peptides on the surface is spatially ordered, the sequence of every peptide is readily known. Replace each amino acid in the 9 amino acid peptide with each of the 20 possible amino acids to identify the amino acid position within this peptide that is essential to form the appropriate peptide-MHCI complex did. FIG. 8 shows all 20 amino acids in one row, grouped by feature, by amino acid one letter code: AFILMVWPGSYCQTNRKHDE. Amino acids A, F, I, L, M, V, W and P are nonpolar amino acids. Amino acids G, S, Y, C, Q, T, N are polar amino acids. Amino acids R, K and H are basic amino acids. Amino acids D and E are acidic amino acids.

Example 4: Linker with negatively charged monomer
[0198] Previously, several linkers were evaluated for the binding of peptides to the array surface to optimized peptide-MHC aggregates on the surface of the aggregate. From that test, a linker consisting of 3-5 HEX moieties (each part containing 6 aminohexanoic acid) is based on the highest signal and low background criteria after detection of pMHC complex with HLA-specific antibodies. Selected as a preferred linker.

  [0199] Continued studies used HLA2-specific peptides well-characterized as biologically relevant peptides that are not only potent HLA2-binding agents but also have potent immunogenic effects. Array experiments were included. When using 15 of these peptides as positive “golden reference” controls, only 7 of these peptides showed the formation of pMHC complexes on the surface of the present disclosure using the preferred HEX linker. I understood.

  [0200] Analysis of substitution plots showing the effect of all 20 amino acids and deletions at each position of the control peptide on pMHC formation was negatively charged at multiple positions for a portion of 7 HLA2-binding peptides The preference of amino acids aspartic acid (D) and glutamic acid (E) was clarified. Without wishing to be bound by theory, the surface can have an excess of positive charge, and the preferred negative charge on the peptide can play a charge-compensating role.

[0201] In order to investigate the effect of charge, various linkers listed below with both negative and positive charges were introduced into the compositions of the present disclosure:
1. Surface-5HEX-peptide2. 2. Surface-HEX-E-3HEX-peptide 3. Surface-HEX-D-3HEX-peptide 4. Surface-HEX-K-3HEX-peptide Surface-2 HEX-E-HEX-peptide 6. Surface-3HEX-GluB-peptide (GluB = t-butyl protected glutamic acid linked via side chain)

  [0202] Linker 1 contains a 5 hexane (HEX) moiety. Linkers 2-4 have a negatively charged glutamic acid (E) or aspartic acid (D) moiety or a positively charged lysine (K), each separated from the peptide by three HEX moieties. Linker 5 is similar to linker 2, but has only one HEX between E and the peptide. Linker 6 is another negatively charged linker having three HEX moieties connected to the side chain of the amino acid analog GluB having a negatively charged free carboxyl group.

  [0203] Analysis of peptide-MHC aggregate formation for 15 control peptides was negatively charged allowing detection of 11 peptides compared to 7 peptides detected with a HEX-only linker. Showed improved binding of the peptide to which the linker was attached. This improvement was less pronounced for linker 6 with GluB amino acids. In contrast, the use of positively charged linker 4 reduced the number of detectable peptides from 7 to 5.

  [0204] FIGS. 10A and 10B show increased signal intensity observed from peptide-MHC aggregates when a linker incorporating at least one negatively charged monomer, in this example aspartic acid (D), is used. Indicates. The linker tested in FIG. 10B corresponds to linker 3 above.

Claims (14)

  A composition comprising at least one assembly comprising a peptide and a major histocompatibility complex (MHC), wherein the peptide is an essential component of MHC and the peptide is surfaced at its C-terminus via a linker The composition, which binds and the peptide is synthesized on the surface.   The composition according to claim 1, wherein the at least one aggregate is a plurality of aggregates.   Composition according to claim 1 or 2, wherein the MHC comprises a carrier molecule, preferably bovine serum albumin (BSA).   The composition according to any one of claims 1 to 3, wherein the MHC is encoded by a human leukocyte antigen (HLA) gene, and the α chain of MHC is cleaved.   The composition according to claim 3, wherein the α chain does not contain a transmembrane region or a cytoplasmic region.   The composition according to claim 4 or 5, wherein the α-chain does not contain a hinge region. The composition according to claim 4 or 5, wherein the α chain comprises an α ₁ domain, an α ₂ domain, and an α ₃ domain.   8. A method of making a composition according to any one of claims 1-7, wherein a surface comprising at least one peptide and an MHC composition produce at least one peptide-MHC aggregate. The peptide is an essential component of MHC, wherein the peptide is bound to the surface at its C-terminus via a linker, and the peptide is synthesized on the surface. Said method. Prior to contacting the surface, the MHC composition is
(A) incubating the MHC composition at 4 ° C. overnight;
(B) separating the precipitate from the MHC composition; and (c) recovering the precipitate-free MHC composition for contact with the surface;
9. The method of claim 8, wherein the method is made by a method comprising:   Use of a composition according to any one of claims 1 to 7 for the identification of one or more peptide antigens that are immunodominant when presented in vivo by an MHC.   The use according to claim 10, wherein the peptide antigen is a cancer antigen.   A vaccine comprising the cancer antigen according to claim 10 or 11.   The cancer antigen according to claim 10 or 11, a sequence complementary to the cancer antigen, an antibody that specifically binds to the cancer antigen, or a cancer antigen for the manufacture of a medicament that enhances or induces an immune response Use of a chimeric antigen receptor that specifically binds to. (A) contacting at least one T cell with the composition;
(B) detecting, upon activation, at least one molecule secreted from at least one T cell; and
(C) imaging at least one T cell;
Use of a composition according to any one of claims 1 to 7 for the identification of at least one T cell stimulated by at least one peptide of the composition comprising: